Assessing the antioxidant activity of melanoidins from coffee brews by different antioxidant methods

Antioxidant activity of instant coffees produced from the same green coffee beans roasted at three different degrees was analyzed. Coffee melanoidins were obtained by ultrafiltration (10 kDa cutoff) and subsequent diafiltration. Pure melanoidins were isolated from melanoidins after overnight incubation in 2 M NaCl and then ultrafiltered. Filtrates, corresponding to the low molecular weight (LMW) fraction noncovalently linked to the melanoidin skeleton, were also preserved. Antioxidant activity of coffee brews (CB), melanoidins (M), pure melanoidins (PM), and bounded melanoidin compounds (BMC) were tested using the 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and ferric reducing power (FRAP) methods. The correlation between the different methods was studied. The higher contribution of melanoidins to the total antioxidant activity of coffees was shown to be caused by the LMW compounds linked noncovalently to the melanoidin skeleton, as data from BMC confirmed. CB, M, and BMC fractions exert the highest antioxidant activity in aqueous media, whereas PM was not dependent on the reaction media. The highest correlation was found between DPPH and FRAP methods.     

Unraveling the contribution of melanoidins to the antioxidant activity of coffee brews

Instant coffees produced from the same green coffee beans were supplied from a company in different roasting degrees, light, medium, and dark. Melanoidins were obtained by ultrafiltration (10 kDa) and subsequent diafiltration. Pure melanoidins were isolated from melanoidins after overnight incubation in 2 M NaCl. The antioxidant activities of instant coffees, melanoidins, and pure melanoidins were tested using the conjugated diene formation from a 2,2'-azobis(2-amidinopropane) dihydrochloride-induced linoleic acid oxidation in an aqueous system. No significant differences were found between melanoidins and pure melanoidins with different roasting degrees. Therefore, the contribution of the pure melanoidin fraction to the total antioxidant activity of melanoidins was significantly lower. More than 50% of the antioxidant activity of melanoidins is due to low molecular weight compounds linked non-covalently to the melanoidin skeleton. A new concept of the overall antioxidant properties of food melanoidins is described, where chelating ability toward low molecular weight antioxidant compounds is connected to the stabilization of these compounds involved in the shelf life of the product.     

Effect of in vitro enzymatic digestion on antioxidant activity of coffee melanoidins and fractions

Traditionally antioxidant activity of melanoidins has only been evaluated in food for implication in shelf life but gastrointestinal digestion is necessary to study their potential bioactivity. In addition, the biological fate of melanoidins has been stressed during the past decade since they did not behave as inert substances. In the present paper a soluble coffee melanoidin isolated from brewed coffee after ultrafiltration with a 10 kDa cutoff membrane was treated ionically and enzymatically collecting the respective high and low molecular weight fractions. Antioxidant activity of these fractions was evaluated with five well-described assays (DPPH, ABTS, ORAC, HOSC, and FRAP) that were previously setup in a plate reader based automatized analysis. Low molecular weight compounds released from melanoidin after gastrointestinal digestion exerted the highest antioxidant activity, even higher than compounds bound ionically to melanoidins. Gastrointestinal digestion is able to modify coffee melanoidins to some extent, as hypothesized from their absolute antioxidant activities. Two options are plausible: by modifying/releasing the ionically bound compounds and/or by genesis of new more active structures from the melanoidin skeleton after enzymatic treatment.     

High molecular weight melanoidins from coffee brew

The composition of high molecular weight (HMw) coffee melanoidin populations, obtained after ethanol precipitation, was studied. The specific extinction coefficient (K(mix)) at 280, 325, 405 nm, sugar composition, phenolic group content, nitrogen content, amino acid composition, and non-protein nitrogen (NPN) content were investigated. Results show that most HMw coffee melanoidins are soluble at high ethanol concentrations. The amino acid composition of the HMw fractions was similar, while 17% (w/w) of the nitrogen was NPN, probably originating from degraded amino acids/proteins and now part of melanoidins. A strong correlation between the melanoidin content, the NPN, and protein content was found. It was concluded that proteins are incorporated into the melanoidins and that the degree of chemical modification, for example, by phenolic groups, determines the solubility of melanoidins in ethanol. Although the existence of covalent interaction between melanoidins and polysaccharides were not proven in this study, the findings suggest that especially arabinogalactan is likely involved in melanoidin formation. Finally, phenolic groups were present in the HMw fraction of coffee, and a correlation was found with the melanoidin concentration.     

Low molecular weight melanoidins in coffee brew

Analysis of low molecular weight (LMw) coffee brew melanoidins is challenging due to the presence of many non-melanoidin components that complicate analysis. This study focused on the isolation of LMw coffee brew melanoidins by separation of melanoidins from non-melanoidin components that are present in LMw coffee brew material. LMw coffee fractions differing in polarity were obtained by reversed-phase solid phase extraction and their melanoidin, sugar, nitrogen, caffeine, trigonelline, 5-caffeoylquinic acid, quinic acid, caffeic acid, and phenolic groups contents were determined. The sugar composition, the charge properties, and the absorbance at various wavelengths were investigated as well. The majority of the LMw melanoidins were found to have an apolar character, whereas most non-melanoidins have a polar character. The three isolated melanoidin-rich fractions represented 56% of the LMw coffee melanoidins and were free from non-melanoidin components. Spectroscopic analysis revealed that the melanoidins isolated showed similar features as high molecular weight coffee melanoidins. All three melanoidin fractions contained approximately 3% nitrogen, indicating the presence of incorporated amino acids or proteins. Surprisingly, glucose was the main sugar present in these melanoidins, and it was reasoned that sucrose is the most likely source for this glucose within the melanoidin structure. It was also found that LMw melanoidins exposed a negative charge, and this negative charge was inversely proportional to the apolar character of the melanoidins. Phenolic group levels as high as 47% were found, which could be explained by the incorporation of chlorogenic acids in these melanoidins.     

Chemical interactions between odor-active thiols and melanoidins involved in the aroma staling of coffee beverages.

Comparative aroma dilution analyses of the headspaces of aqueous solutions containing either the total volatiles isolated from a fresh coffee brew, or these volatiles remixed with the melanoidins isolated from coffee brew, revealed a drastic decrease in the concentrations of the odorous thiols 2-furfurylthiol, 3-methyl-2-butenthiol, 3-mercapto-3-methylbutyl formate, 2-methyl-3-furanthiol, and methanethiol when melanoidins were present. Among these thiols, 2-furfurylthiol was affected the most: e.g., its concentration decreased by a factor of 16 upon addition of melanoidins. This was accompanied by a decrease in the overall roasty-sulfury aroma. Quantitations performed by means of stable isotope dilution assays confirmed the rapid loss of all thiols with increasing time while keeping the coffee brew warm in a thermos flask. Using [2H2]-2-furfurylthiol as an example, [2H]-NMR and LC/MS spectroscopy gave strong evidence that thiols are covalently bound to the coffee melanoidins via Maillard-derived pyrazinium compounds formed as oxidation products of 1,4-bis-(5-amino-5-carboxy-1-pentyl)pyrazinium radical cations (CROSSPY). Using synthetic 1,4-diethyl diquaternary pyrazinium ions and 2-furfurylthiol, it was shown that 2-(2-furyl)methylthio-1,4-dihydro-pyrazines, bis[2-(2-furyl)methylthio]-1,4-dihydro-pyrazines, and 2-(2-furyl)methylthio-hydroxy-1,4-dihydro-pyrazines were formed as the primary reaction products. Similar results were obtained for models in which either 1,4-diethyl diquaternary pyrazinium ions were substituted by Nalpha-acetyl-L-lysine/glycolaldehyde, or the 2-furfurylthiol by 2-methyl-3-furanthiol and 3-mercapto-3-methylbutyl formate. On the basis of these results it can be concluded that the CROSSPY-derived pyrazinium intermediates are involved in the rapid covalent binding of odorous thiols to melanoidins, and, consequently, are responsible for the decrease in the sulfury-roasty odor quality observed shortly after preparation of the coffee brew.     

Angiotensin-I converting enzyme inhibitory activity of coffee melanoidins

Melanoidins formed at the last stage of the Maillard reaction have been pointed out to possess certain functional properties. Potential antihypertensive activity of food melanoidins (coffee, beer, and sweet-wine) has been evaluated according to in-vitro ACE-inhibitory activity. Precision of the assay (3.2% of coefficient of variation, n = 10) for melanoidins is similar to those reported of well-known antihypertensive peptides. Assay was applied on food melanoidins obtained from coffee (three roasting degrees), beer, and sweet-wine. All samples showed in-vitro ACE-inhibitory activity. The activity in coffee melanoidins was significantly higher at more severe heating conditions. These experiments demonstrate that food melanoidins could inhibit ACE activity. In-vitro ACE-inhibitory activity of coffee melanoidins is likely located within the melanoidin structure. But ACE-inhibitory activity is also partly due to the low-molecular-weight compound nonchemically bound to the melanoidin structure, then melanoidins can act as carrier-protecting agents. These compounds could be naturally phenolic compounds present in the green beans or intermediary Maillard reaction products with antihypertensive activity.     

Incorporation of chlorogenic acids in coffee brew melanoidins

The incorporation of chlorogenic acids (CGAs) and their subunits quinic and caffeic acids (QA and CA) in coffee brew melanoidins was studied. Fractions with different molecular weights, ionic charges, and ethanol solubilities were isolated from coffee brew. Fractions were saponified, and the released QA and CA were quantified. For all melanoidin fractions, it was found that more QA than CA was released. QA levels correlated with melanoidin levels, indicating that QA is incorporated in melanoidins. The QA level was correlated with increasing ionic charge of the melanoidin populations, suggesting that QA may contribute to the negative charge and consequently is, most likely, not linked via its carboxyl group. The QA level correlated with the phenolic acid group level, as determined by Folin-Ciocalteu, indicating that QA was incorporated to a similar extent as the polyphenolic moiety from CGA. The QA and CA released from brew fractions by enzymes confirmed the incorporation of intact CGAs. Intact CGAs are proposed to be incorporated in melanoidins upon roasting via CA through mainly nonester linkages. This complex can be written as Mel=CA-QA, in which Mel represents the melanoidin backbone, =CA represents CA nonester-linked to the melanoidin backbone, and -QA represents QA ester-linked to CA. Additionally, a total of 12% of QA was identified in coffee brew, whereas only 6% was reported in the literature so far. The relevance of the additional QA on coffee brew stability is discussed.     

Roasting effects on formation mechanisms of coffee brew melanoidins

The effect of the roasting degree on coffee brew melanoidin properties and formation mechanisms was studied. Coffee brew fractions differing in molecular weight (Mw) were isolated from green and light-, medium-, and dark-roasted coffee beans. Isolated fractions were characterized for their melanoidin, nitrogen, protein, phenolic groups, chlorogenic acid, quinic acid, caffeic acid, and sugar content. It was found that the melanoidin level in all fractions correlated with both the nitrogen and the protein content. The melanoidin level also correlated with the phenolic groups' level and ester-linked quinic acid level. It was concluded that proteins and chlorogenic acids should be primarily involved in melanoidin formation. Initial roasting, from green to light-roasted beans, especially led to the formation of intermediate Mw (IMw) melanoidins when compared to high Mw (HMw) melanoidins. Indications were found that this IMw melanoidin formation is mainly due to Maillard reactions and chlorogenic acid incorporation reactions between chlorogenic acids, sucrose, and amino acids/protein fragments. Additionally, it was found that prolonged roasting predominantly led to formation melanoidins with a high Mw. Furthermore, arabinogalactans seem to be relatively more involved in melanoidin formation than galactomannans. It was hypothesized that chromophores may be formed or attached through the arabinose moiety of arabinogalactan proteins (AGP). Finally, it could be concluded that galactomannans are continuously incorporated in AGP-melanoidins upon roasting.     

Model studies on the influence of coffee melanoidins on flavor volatiles of coffee beverages

Addition of the total melanoidin fraction isolated by water extraction from medium-roasted coffee powder to a model solution containing a set of 25 aroma compounds mimicking the aroma of a coffee brew reduced, in particular, the intensity of the roasty, sulfury aroma quality. Model studies performed by static headspace analysis revealed that especially three well-known coffee odorants, that is, 2-furfurylthiol (FFT), 3-methyl-2-butene-1-thiol, and 3-mercapto-3-methylbutyl formate, were significantly reduced in the headspace above an aqueous model solution when melanoidins were added. In particular, the low molecular weight melanoidins (1500-3000 Da) led to the most significant decrease in FFT. In contrast, for example, aldehydes remained unaffected by melanoidin addition.     

Influence of coffee roasting on the incorporation of phenolic compounds into melanoidins and their relationship with antioxidant activity of the brew

In the present study, the influence of coffee roasting on free and melanoidin-bound phenolic compounds and their relationship with the brews' antioxidant activity (AA), evaluated by TRAP, TEAC, and TRAP, were investigated. Changes in the relative content of free chlorogenic acids (CGA), free lactones, and melanoidin-bound phenolic acids during roasting indicate that phenolic compounds were incorporated into melanoidins mainly at early stages of the process, being thereafter partly oxidized to dihydrocaffeic acid, and degraded. Although less than 1% of CGA in green coffee was incorporated into melanoidins during roasting, the relative content of melanoidin-bound phenolic acids increased significantly during this process, reaching up to 29% of total phenolic compounds in brews from dark roasted coffees. Regardless of the AA assay used and considering all roasting degrees, the overall contribution of CGA to the AA of the whole brews was higher than that of melanoidin-bound phenolic compounds. It was estimated that the latter compounds contributed to 25-47% of the AA, depending on the assay used.     

Arabinogalactan proteins are incorporated in negatively charged coffee brew melanoidins

The charge properties of melanoidins in high molecular weight (HMw) coffee brew fractions, isolated by diafiltration and membrane dialysis, were studied. Ion exchange chromatography experiments with the HMw fractions showed that coffee brew melanoidins were negatively charged whereas these molecules did not expose any positive charge at the pH of coffee brew. Fractions with different ionic charges were isolated and subsequently characterized by means of the specific extinction coefficient (K(mix 405nm)), sugar composition, phenolic group content, nitrogen content, and the arabinogalactan protein (AGP) specific Yariv gel-diffusion assay. The isolated fractions were different in composition and AGP was found to be present in one of the HMw fractions. The AGP accounted for 6% of the coffee brew dry matter and had a moderate negative charge, probably caused by the presence of uronic acids. As the fraction that precipitated with Yariv was brown (K(mix 405nm) = 1.2), compared to a white color in the green bean, it was concluded that these AGPs had undergone Maillard reaction resulting in an AGP-melanoidin complex. The presence of mannose (presumably from galactomannan) indicates the incorporation of galactomannans in the AGP-melanoidin complex. As the uronic acid content in the more negatively charged melanoidin-rich, AGP-poor HMw fractions decreased, it was hypothesized that acidic groups are formed or incorporated during melanoidin formation.     

High molecular weight coffee melanoidins are inhibitors for matrix metalloproteases

High molecular (above 10 kDa) melanoidins isolated from coffee beans of varying roasting degree were found to be efficient inhibitors for the zinc-containing matrix metalloproteases MMP-1, MMP-2, and MMP-9 with IC(50) values ranging between 0.2 and 1.1 mg/mL in vitro. The inhibitory potential increased with roasting degree. No or only slight inhibition of other zinc-containing peptidases closely related to MMPs, namely, Clostridium histolyticum collagenase and angiotensin converting enzyme, was found, indicating specific structural features of melanoidins to be responsible for the interaction with MMPs. A continuous increase on the apparent molecular weight of melanoidins as well as incorporation of phenolic substances into the melanoidin structure with progress of roasting was observed, concomitant with a significant increase in the carbon/nitrogen of the melanoidins. This suggests that the melanoidins are mainly formed by incorporation of carbohydrates and phenolic compounds onto a proteinaceous backbone. As MMP-1, MMP-2, and MMP-9 play a pivotal role in pathogenesis of colorectal cancer, studies on possible physiological effects of melanoidins are mandatory.     

Chemical composition and antioxidant properties of clove leaf essential oil

The antioxidant activity of a commercial rectified clove leaf essential oil (Eugenia caryophyllus) and its main constituent eugenol was tested. This essential oil comprises in total 23 identified constituents, among them eugenol (76.8%), followed by beta-caryophyllene (17.4%), alpha-humulene (2.1%), and eugenyl acetate (1.2%) as the main components. The essential oil from clove demonstrated scavenging activity against the 2,2-diphenyl-1-picryl hydracyl (DPPH) radical at concentrations lower than the concentrations of eugenol, butylated hydroxytoluene (BHT), and butylated hydroxyanisole (BHA). This essential oil also showed a significant inhibitory effect against hydroxyl radicals and acted as an iron chelator. With respect to the lipid peroxidation, the inhibitory activity of clove oil determined using a linoleic acid emulsion system indicated a higher antioxidant activity than the standard BHT.     

In vitro antioxidant activity of coffee compounds and their metabolites

In this paper we report the antioxidant activity of different compounds which are present in coffee or are produced as a result of the metabolism of this beverage. In vitro methods such as the ABTS*+ [ABTS = 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)] decolorization assay and the oxygen radical absorbance capacity assay (ORAC) were used to assess the capacity of coffee compounds to scavenge free radicals. The importance of caffeine metabolites and colonic metabolites in the overall antioxidant activity associated with coffee consumption is shown. Colonic metabolites such as m-coumaric acid and dihydroferulic acid showed high antioxidant activity. The ability of these compounds to protect human low-density lipoprotein (LDL) oxidation by copper and 2,2'-azobis(2-amidinopropane) dihydrochloride was also explored. 1-Methyluric acid was particularly effective at inhibiting LDL oxidative modification. Different experiments showed that this caffeine metabolite is not incorporated into LDL particles. However, at physiologically relevant concentrations, it was able to delay for more than 13 h LDL oxidation by copper.     

Chemical characterization of galactomannans and arabinogalactans from two arabica coffee infusions as affected by the degree of roast

Galactomannans and arabinogalactans compose almost exclusively the polysaccharide fraction of roasted coffee infusions. To increase the knowledge about the effect of the degree of roast (DR) in the amount and chemical structure of the galactomannans and arabinogalactans, two arabica coffees of different geographical origins (Costa Rica and Brazil) were roasted for three degrees of roast (DRs 4.7-5.0, 8.7, and 10% of dry weight loss of green coffee beans, on a dry basis). The high molecular weight material was extracted with hot water and dialyzed (molecular weight cutoff >12 kDa), and the material was separated in three cold-water-soluble fractions by graded addition of ethanol. The degree of polymerization and the degree of branching of the galactomannans decreased with the increase of the DR. As the DR increased, less branched arabinogalactans were extracted. The relative amount of terminally linked arabinosyl residues of the arabinogalactans decreased with the increase in DR, and the terminally linked galactosyl residues increased. Also, the size of the arabinosyl side chains of the arabinogalactans decreased with the increase in DR.     

Chemical characterization of the high molecular weight material extracted with hot water from green and roasted arabica coffee

The polysaccharides present in coffee infusions are known to contribute to the organoleptic characteristics of the drink, such as the creamy sensation perceived in the mouth known as "body", the release of aroma substances, and the stability of espresso coffee foam. To increase the knowledge about the origin, composition, and structure of the polysaccharide fraction, the high molecular weight material (HMWM) was extracted with hot water from two green and roasted ground arabica coffees: Costa Rica (wet processed) and Brazil (dry processed). The polysaccharides present in the green coffees HMWM were arabinogalactans (62%), galactomannans (24%), and glucans, and those found in roasted coffees were galactomannans (69%) and arabinogalactans (28%). The polysaccharides of the HMWM of the roasted coffees were less branched than those of the green coffees. The major green coffee proteins had molecular weights of 58 and 38 kDa, and the 58 kDa protein had two subunits, of 38 and 20 kDa, possibly linked by disulfide bonds. The protein fraction obtained from roasted coffees had only a defined band with < or =14 kDa and a diffuse band with >200 kDa. The majority of the galactomannans were precipitated with solutions of 50% ethanol, and the size-exclusion chromatography of the roasted fractions showed coelution of polysaccharides, proteins, phenolics, and brown compounds. The use of strong hydrogen and hydrophobic dissociation conditions allowed us to conclude that the phenolics and brown compounds were linked by covalent bonds to the polymeric material.     

Effect of roasting on the antioxidant activity of coffee brews

Colombian Arabica coffee beans were roasted to give light, medium, and dark samples. Their aqueous extracts were analyzed by gel filtration chromatography, UV-visible spectrophotometry, capillary electrophoresis, and the ABTS(*)(+) assay. A progressive decrease in antioxidant activity (associated mainly with chlorogenic acids in the green beans) with degree of roasting was observed with the simultaneous generation of high (HMM) and low molecular mass (LMM) compounds possessing antioxidant activity. Maximum antioxidant activity was observed for the medium-roasted coffee; the dark coffee had a lower antioxidant activity despite the increase in color. Analysis of the gel filtration chromatography fractions showed that the LMM fraction made a greater contribution to total antioxidant activity than the HMM components.     

Antioxidant properties of ready-to-drink coffee brews

The influence of some technological variables on the changes of the antioxidant capacity of ready-to-drink coffee brews was investigated. Results showed that, depending on the roasting degree as well as on the packaging conditions adopted, redox reactions, which can take place during storage, are responsible for significant changes in the overall antioxidant capacity of the product. In particular, the redox potential of air-packaged coffee brews, obtained from light- and medium-roasted beans, showed maximum values after 2 days of storage, which corresponded to a minimum in the chain-breaking activity, while, in the case of the dark-roasted sample packaged under ordinary atmosphere, both the redox potential and the chain-breaking activity showed a maximum around 2-3 days of storage. In contrast, in the absence of oxygen, the coffee brews maintained the initial reducing properties over all the storage time, although the radical-scavenging activity values changed in a way very similar to that of the air-packaged sample. These results suggested that the changes in the antioxidant properties of the coffee brews may be attributed to a further development of the Maillard reaction during storage.     

Antioxidant properties of roasted coffee residues

The antioxidant activity of roasted coffee residues was evaluated. Extraction with four solvents (water, methanol, ethanol, and n-hexane) showed that water extracts of roasted coffee residues (WERCR) produced higher yields and gave better protection for lipid peroxidation. WERCR showed a remarkable protective effect on oxidative damage of protein. In addition, WERCR showed scavenging of free radicals as well as the reducing ability and to bind ferrous ions, indicating that WERCR acts as both primary and secondary antioxidants. The HPLC analyses showed that phenolic acids (chlorogenic acid and caffeic acid) and nonphenolic compounds [caffeine, trigonelline, nicotinic acid, and 5-(hydroxymethyl)furfuraldehyde] remained in roasted coffee residues. These compounds showed a protective effect on a liposome model system. The concentrations of flavonoids and polyphenolic compounds in roasted coffee residues were 8,400 and 20,400 ppm, respectively. In addition, the Maillard reaction products (MRPs) remaining in roasted coffee residues were believed to show antioxidant activity. These data indicate that roasted coffee residues have excellent potential for use as a natural antioxidant source because the antioxidant compounds remained in roasted coffee residues.